Phosphorylated Tri-Block Copolymers with Anti-Microbial Properties

ABSTRACT

The disclosure provides products and methods of treating diseases and disorders involving microbial pathogens, such as intestinal microbial pathogens, e.g., Pseudomonas aeruginosa, by administering an effective amount of a phosphorylated polyethylene glycol compound of defined structural organization. Those diseases and disorders characterized by an epithelium attacked by a microbial pathogen are contemplated, including gastrointestinal infections and inflammation, e.g., treatment of intestinal or esophageal anastomosis or treatment or suppression of anastomotic leakage.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.R01GM062344-15 awarded by the National Institutes of Health and underContract No. DE-AC002-06CH11357 awarded by the U.S. Department ofEnergy. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of the disclosure, aSequence Listing in computer-readable form which is incorporated byreference in its entirety and identified as follows: Filename:51283A_Seqlisting.txt; 781 bytes, created Sep. 25, 2017.

FIELD

The disclosure relates generally to the fields of medical treatment,prevention or suppression of epithelial diseases and disorders includinginfections and inflammation.

BACKGROUND

The promiscuous use of antibiotics had led to the emergence ofantibiotic resistance at an unprecedented pace and continues to placepatients at risk for life-threatening infections following majorsurgery. Many if not most of the pathogens that cause these infectionsuse the intestinal tract as their primary site of colonization. Althoughsurgeons routinely decontaminate the intestinal tract with antibioticsprior to surgery to prevent infection, this practice carries theunintended consequence of causing antibiotic resistance. Furthermore,overuse of antibiotics destroys the microbiome that normally protectsagainst high risk pathogens. A more evolutionarily stable strategy tothis problem would be to develop compounds that suppress pathogenvirulence without affecting the growth of the pathogenic organisms,thereby preserving the microbiome. In this manner, bacterialpathogenicity could be contained and the colonization resistance of thenormally protective microbiota preserved.

Phosphate is a key universal cue for bacteria to either enhance theirvirulence, for example when local phosphate is scarce, or to suppressvirulence when phosphate is abundant. Phosphate can become depleted inthe mammalian gut following physiologic stress and serves as a majortrigger for colonizing bacteria to express virulence and invasiveness.This process cannot be reversed with oral or intravenous inorganicphosphate, as phosphate is nearly completely absorbed in the proximalsmall intestine.

One obstacle to using commercially available PEG 15-20 (Sigma) is thatPEG 15-20 is not a pure tri-block polymer but rather a mixture ofpolymers of varying molecular weights. This situation limits the abilityto further interrogate the molecular mechanisms by which the polymersprotect both in vitro and in vivo, and the limited content of phosphateinhibits the opportunity to further improve the efficacy of thepolymers.

In view of the foregoing observations, it is apparent that needscontinue to exist in the art for products and methods useful intreating, preventing or suppressing diseases and disorders characterizedby a pathogenic microbial attack on an epithelium.

SUMMARY

The disclosure provides materials and methods for treating, preventingor suppressing diseases and conditions associated with pathogenicmicrobe-mediated epithelial diseases or disorders such asgastrointestinal infections or inflammation, or gastrointestinalanastomoses or anastomotic leaks, such as esophageal or intestinalanastomoses or anastomotic leaks. The materials for use in suchcircumstances are phosphorylated polyethylene glycol compounds of adefined structure, such as an A-B-A triblock copolymer structure(ABA-PEG-Pi). Notably, the ABA-PEG-Pi materials of the disclosurecomprise a hydrophobic core such as a diphenylmethyl moiety and thematerials exhibit a substantially similar molecular weight wherein about80%, 90%, 95%, 96%, 97% 98%, 99%, 99.5% or 99.9% of the ABA-PEG-Pimolecules have the same molecular weight (plus or minus 5% or 10%). Theparticular structure of the ABA-PEG-Pi and the relatively constantstructure result in effects on epithelial cell diseases and disordersmediated by obligate or opportunistic microbial pathogens.

One aspect of the disclosure is drawn to a triblock copolymercomprising: (a) a hydrophobic core; and (b) at least two polyethyleneglycol chains wherein at least one polyethylene glycol chain is aphosphorylated polyethylene glycol comprising more than two phosphategroups. The hydrophobic core is a “B” block copolymer and the PEG chainsare generally considered “A” block copolymers using the triblockcopolymer terminology of the disclosure. It is recognized that triblockcopolymers can be indicated as having an A-B-A or an A-B-A′ triblockcopolymer structure depending on whether the at least two PEG chains arethe same or not. In some embodiments, at least two polyethylene glycolchains are phosphorylated polyethylene glycol chains comprising morethan two phosphate groups. In some embodiments, the hydrophobic core isa carbocyclic or heterocyclic ring, including embodiments wherein thering is aromatic, such as a single ring or a plurality of rings. In someembodiments, the hydrophobic core is a diphenylmethyl moiety. In someembodiments, the hydrophobic core is a4,4′-(propane-2,2-diyl)diphenolate salt. In some embodiments, thecopolymer has a molecular weight of at least 8,000 daltons, at least12,000 daltons, at least 15,000 daltons, at least 16,000 daltons, atleast 20,000 daltons, or is between 15,000-20,000 daltons. In someembodiments, the copolymer is in solution. In some embodiments, thedispersity (Ð) of the triblock copolymer disclosed herein is less thanor equal to 1.10. In some embodiments, the triblock copolymer is aphosphorylated form of ABA-PEG-PGly or ABA-PEG-PEEGE.

Another aspect of the disclosure is directed to a method of producingthe triblock copolymer comprising (a) covalently attaching at least twopolyethylene glycol chains to a hydrophobic core comprising acarbocyclic or heterocyclic ring; and (b) covalently attaching at leasttwo phosphate groups to at least one polyethylene glycol chain. In someembodiments, at least two polyethylene glycol chains are each covalentlyattached to at least two phosphate groups.

Yet another aspect of the disclosure is a method of treating anastomosiscomprising administering a therapeutically effective amount of acomposition comprising a triblock copolymer disclosed herein to asubject in need. In some embodiments, the triblock copolymer has amolecular weight of at least 8,000 daltons, 12,000 daltons, 15,000daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000daltons.

Still another aspect of the disclosure is a method of treatinganastomotic leakage comprising administering a therapeutically effectiveamount of a composition comprising a triblock copolymer disclosed hereinto a subject in need. In some embodiments, the triblock copolymer has amolecular weight of at least 8,000 daltons, 12,000 daltons, 15,000daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000daltons.

In another aspect, the disclosure provides a method of preventinganastomotic leakage comprising administering an effective amount of acomposition comprising a triblock copolymer disclosed herein to asubject at risk of anastomotic leakage. In some embodiments, thetriblock copolymer has a molecular weight of at least 8,000 daltons,12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, orbetween 15,000-20,000 daltons.

Yet another aspect of the disclosure is directed to a method ofsuppressing anastomotic leakage comprising administering an effectiveamount of a composition comprising a triblock copolymer disclosed hereinto a subject at risk of anastomotic leakage. In some embodiments, thetriblock copolymer has a molecular weight of at least 8,000 daltons,12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons, orbetween 15,000-20,000 daltons.

Another aspect of the disclosure is drawn to a method of inhibiting,e.g., preventing, microbial virulence, such as the virulence ofPseudomonas aeruginosa or the virulence of Enterococcus faecalis.Virulence is herein defined as the relative ability of a microorganism,such as P. aeruginosa or E. faecalis, to cause disease, which is ameasure of the degree of pathogenicity. Stated alternatively, virulencereflects the capability of a microorganism to cause disease. Virulencein P. aeruginosa can be characterized by the P2 (or virulent) phenotype,wherein P. aeruginosa one or more of the following features: producestoxin(s), exhibits swarming motility, activates expression of thequorum-sensing system (QS), has elevated expression of PstS, and/orincreases production of pyocyanin and/or pyoverdin. Virulence in E.faecalis can be characterized by increased collagenase production.Accordingly, the disclosure provides a method of inhibiting microbialvirulence in the gastrointestinal tract comprising administering aneffective amount of a composition comprising a triblock copolymer asdisclosed herein to a subject at risk of inducing microbial virulence.In some embodiments, the triblock copolymer has a molecular weight of atleast 8,000 daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons,20,000 daltons, or between 15,000-20,000 daltons. In some embodiments,the microbe is Pseudomonas aeruginosa. In some embodiments, the microbeis Enterococcus faecalis.

The methods of inhibiting microbial virulence are related to anotheraspect of the disclosure providing a method of treating agastrointestinal microbe capable of developing a virulent phenotypecomprising administering an effective amount of a composition comprisinga triblock copolymer as disclosed herein to a subject comprising thegastrointestinal microbe. In some embodiments, the triblock copolymerhas a molecular weight of at least 8,000 daltons, 12,000 daltons, 15,000daltons, 16,000 daltons, 20,000 daltons, or between 15,000-20,000daltons. In some embodiments, the gastrointestinal microbe capable ofdeveloping a virulent phenotype is Pseudomonas aeruginosa. In someembodiments, the gastrointestinal microbe capable of developing avirulent phenotype is Enterococcus faecalis.

Other features and advantages of the disclosure will become apparentfrom the following detailed description, including the drawing. Itshould be understood, however, that the detailed description and thespecific examples, while indicating embodiments, are provided forillustration only, because various changes and modifications within thespirit and scope of the disclosure will become apparent to those skilledin the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 . Reaction scheme for the synthesis of ABA-Pi-PEG blockcopolymers.

FIG. 2 . ¹H-NMR spectra of (A) ABA-PEG20k-E18, (B) ABA-PEG20k-G20, (C)ABA-PEG20k-Pi20, (D) ³¹P-NMR spectrum of ABA-PEG20k-Pi20, and (E)Titration curve of ABA-PEG20k-Pi20 with NaOH solution. 20 k is thedesigned molecular weight of PEG block; E18 means the designed repeatingunits of EEGE block is 18; G20 means the designed repeating units ofGlycerol is 20. Pi20 means the designed repeating units of thephosphorylated Glycerol block is 20.

FIG. 3 . ABA-Pi-PEGs significantly decrease PstS expression in P.aeruginosa. PstS expression in MPAO1/pstS-EGFP (A), and ΔPvdD/pstS-EGFP(B). n=3 per group, *p<0.01. Columns represent average values, and errorbars—standard deviations.

FIG. 4 . ABA-Pi-PEGs significantly decrease pyocyanin production in P.aeruginosa. (A) Production of pyocyanin in P. aeruginosa MAPO1-P2 grownin phosphate/iron-limited medium DCM-Pi0.1, phosphate-limited mediumDCM-Pi0.1+Fe³⁺, 2 and phosphate-limited/iron-enriched media supplementedwith 1 mM phosphorylated and non-phosphorylated polymers. (B) Productionof pyocyanin in MPAO1-P1 in TSB supplemented with 0.2 mM U-50,488 in thepresence or absence of phosphorylated and non-phosphorylated polymers.n=3 per group, *p<0.01. Columns represent average values, and errorbars-standard deviations.

FIG. 5 . ABA-Pi-PEGs and ABA-PEGs significantly decrease pyoverdinproduction in P. aeruginosa. n=3 per group, *p<0.01. Columns representaverage values, and error bars—standard deviations.

FIG. 6 . Effect of each of the three phosphorylated ABA polymers on C.elegans survival. Experiments are performed on C. elegans nematodesfeeding on P. aeruginosa in low nutrient media (0.1×TY) and exposed toopioids (U50,488) as a provocative agent known to enhance P. aeruginosavirulence. Kaplan-Meyer survival curves demonstrate a statisticallysignificant (p<0.05) protective effect of all three polymers at 2 mMconcentration when compared to the no treatment group. Results indicatethat the ABA-PEG20k-Pi20 confers a superior protective effect comparedto the remaining polymers (n=10 worms/plate (treatment group), 3independent runs per group.

FIG. 7 . Composition analysis of PEG15-20 (Sigma) and Pi-PEG15-20. (A)GPC traces of commercial PEG15-20 and its phosphorylated productPi-PEG15-20 in 0.1 M NaNO₃ (25° C., 1.0 ml/minute) indicate thatPEG15-20 is a mixture of homopolymers and copolymers with ABA and ABstructures. (B) Analysis reveals that 16% of PEG15-20 contained ABAstructure, as calculated from the integration area of the GPC curve.

FIG. 8 . GPC traces of (A) ABA-PEG-PEEGE, ABA-PEG-PGly intetrahydrofuran (THF; 35° C., 0.8 ml/minute); (B) ABA-PEG-Pi in 0.1 MNaNO₃ (25° C., 1.0 ml/minute); (C) PEG-PEEGE, PEG-PGly in THF (35° C.,0.8 ml/minute); and (D) PEG-Pi in 0.1 M NaNO₃ (25° C., 1.0 ml/minute).

FIG. 9 . ABA-Pi20-PEG20 k does not inhibit growth of P. aeruginosa.Absorbance at OD_(600 nm) measured in overnight culture of P. aeruginosaMPAO1/psts-EGFP in DCM-Pi0.1 and DCM-Pi0.1 supplemented with 2 mMABA-PEG20k-Pi20. Results present mean data of three biologicalreplicates.

FIG. 10 . Comparison of the chemical structure of phosphate-containingPEG-based block copolymers: ABA-PEG-Pi with the hydrophobic core BPA andPEG-Pi without BPA.

FIG. 11 . The hydrophobic core BPA in ABA-PEG20k-Pi20 significantlycontributes to bacterial coating and its in vivo protection againstlethality. (A) PstS expression. N=3/group, *p<0.001. (B) C. eleganssurvival. N=60/group, p<0.0001 between groups (Long-rank (Mantiel-Cox)test). (C-E) Scanning electron microscopy images of P. aeruginosacultured in different media. The bacteria were first cultured indifferent media for several hours, then immediately after washing withbuffer solution, dried in a critical point dryer, coated with Pt/Pd andimages taken. Arrows indicate pili-like filaments. (C), cultured inphosphate-limited (DCM Pi-0.1 mM) media only, (D) cultured in DCM Pi-0.1mM containing 2 mM PEG20k-Pi20, and (E) cultured in DCM Pi-0.1 mMcontaining 2 mM ABA-PEG20k-Pi20.

FIG. 12 . Schematic illustration of ABA-PEG-Pi block co-polymer and itsfunction suppressing virulence of pathogenic microbes in vitro andattenuation of mortality in vivo, as well as its capacity to coat thesurface of bacteria. The central schematic shows a central hydrophobiccore flanked by PEG blocks on each side, with terminal phosphate blocks.

FIG. 13 . ¹H-NMR spectra of (A) PEG20k-E18; (B) PEG20k-G20; (C)PEG20k-Pi20; and (D) ³¹P-NMR spectrum of PEG20k-Pi20.

FIG. 14 . Effect of phosphorylated ABA polymers on C. elegans survival.Experiments were performed on C. elegans nematodes feeding on P.aeruginosa in low nutrient media (0.1×TY) and exposed to opioids(U50,488) as a provocative agent known to enhance P. aeruginosavirulence. Kaplan-Meyer survival curves demonstrate a statisticallysignificant (p=0.01, Log-rank Mantel-Cox test) protective effect ofABA-PEG20k-Pi20 compared to ABA-PEG10k-Pi10 at 5% concentration. Bothpolymers have significant protective effect compared to the no-PEG group(p<0.0001, Log-rank Mantel-Cox test). Results indicate that theABA-PEG20k-Pi20 confers a superior protective effect compared to theremaining polymers examined in the study (n=10 worms/plate (treatmentgroup), 3 independent runs per group).

FIG. 15 . Strategy for the synthesis of PEG-Pi polymers.

FIG. 16 . Phosphorylated PEG is effective when administered orallythrough drinking water. (A) FIG. 10(A) provides data showing the extentof cell proliferation in cecal crypts of mice provided with drinkingwater lacking a phosphorylated PEG (PC 1-3, i.e., pathogen community,plates 1-3 wherein each plate contained 10 or 15 C. elegans wormsexposed to pathogens), drinking water containing 3% PPi6(hexametaphosphate) (PPi6 1-3 refers to plates 1-3 as described above,but exposed to pathogens from mice drinking 3% PPi6), or drinking watercontaining 1% ABA-PEG20 k (ABA-PEG20k-P120 1-3, wherein 1-3 refers toplates 1-3 as described above exposed to pathogen from mice drinking 1%ABA-PEG20k). All groups are SAH+PC (starvation-positive,antibiotic-positive hepatectomy subjects exposed to the pathogencommunity), POD2 (data from post-operative day 2), and cellproliferation is measured by immunostaining for Ki67. The results revealthat cells proliferate to a height of 71.16±3.055 μm above the cryptbase when drinking water lacking any phosphorylated PEG. In contrast,cells proliferate to a height of 31.04±1.288 μm or a height of45.42±1.403 μm above the cecal crypt base in mice drinking watercontaining 3% PPi6 or 1% ABA-PEG20k, respectively. (B) FIG. 10(B) showscell proliferation relative to cecal crypt depth for mice drinking waterlacking any phosphorylated PEG (PC 1-3), mice drinking water containing3% PPi6 (PPi6 1-3), or mice drinking water containing 1% ABA-PEG20 k(ABA-PEG20k-Pi20 1-3), wherein “1-3” refers to individual mice subjectedto the indicated conditions. The height of proliferating cells relativeto cecal crypt depth were 50.54±1.711 μm for water only, 35.12±1.332 μmfor 3% PPi6, and 39.01±0.909 μm for 1% ABA-PEG20k.

DETAILED DESCRIPTION

A phosphorylated polyethylene glycol compound of high molecular weightallows phosphate to be distributed along the entire gut and into thedistal intestine where microbes such as bacteria are most abundant. Thephosphorylated polyethylene glycol compounds of the disclosure have atriblock copolymer structure of ABA, with “A” referring to anypolyethylene glycol, or derivative thereof, that is at least 8,000daltons, 12,000 daltons, 15,000 daltons, 16,000 daltons, 20,000 daltons,or is between 15,000-20,000 daltons. The “B” component of the triblockstructure is a hydrophobic compound capable of covalent linkage to twoPEG molecules of the disclosure, or derivatives thereof. Exemplaryhydrophobic cores are bisphenol A (BPA) and bisphenol E (BPE).

In discussing the compounds of the disclosure, and compositionscomprising such compounds, the following terminology is used. “ABA”refers to the triblock structural organization of the compounds, withtwo like polymers, e.g., PEG, bracketing a “B” structure that is ahydrophobic core such as any aliphatic, carbocyclic, heterocyclic, oraromatic structure that is hydrophobic, e.g. any of the bisphenols.“PEG” refers to polyethylene glycol, and “PEG-Pi” refers to aphosphorylated polyethylene glycol. “EEGE” is ethoxyethyl glycidyl etherand “PEEGE” is polyethoxyethyl glycidyl ether. As described below, EEGEis de-protected and, once de-protected, EEGE groups become hydroxygroups and the structure is referred to as a polyglycidol, such asABA-PEG-PGly. Compounds identified as ABA-PEG10k-E8, ABA-PEG16k-E12, andABA-PEG20k-E18 refer to triblock copolymers having the ABA structurewith 8 EEGE groups (E8) and PEG groups of 10 k in ABA-PEG8k-E8. ForABA-PEG16k-E12, the compound has the ABA structure with 12 EEGE groups(E12) and PEG groups of 16 k. In like manner, ABA-PEG20k-E18 has an ABAstructure with 18 EEGE groups and PEG groups of 20 k. For compoundsidentified as ABA-PEG10k-G10, ABA-PEG16k-G14 and ABA-PEG20k-G20, “G10”refers to 10 hydroxyl groups created by de-protection of EEGE (the “G”is a reference to the compound as a polyglycidol), while “G14” and “G20”refer to 14 and 20 hydroxyl groups, respectively. Compounds defined asABA-PEG10k-Pi10, ABA-PEG16k-Pi14, and ABA-PEG20k-Pi20 refer to compoundshaving the ABA triblock copolymer structure with 10, 14, or 20phosphoryl groups (e.g., phosphate groups), respectively, resulting fromphosphorylation of a polyglycidol. Consistent with the naming conventionexplained above, PEG10k, PEG16k, and PEG20k refer to PEG groups of 10 k,16 k and 20 k, respectively. It is apparent that the number offunctional groups (e.g., EEGE) ultimately rendered amenable tophosphorylation can vary in the compounds according to the disclosure,and the size of PEG molecules bearing those functional groups can vary,including PEG molecules in a compound totaling at least 8,000 daltons,at least 12,000 daltons, at least 15,000 daltons, at least 16,000daltons, at least 20,000 daltons or between 15,000-20,000 daltons.

The phosphate content of compounds delivered to the intestine isparticularly important for any protective effect because local phosphateconcentrations support bacterial growth while at the same timesuppressing bacterial virulence [28]. The mechanism underlying thiseffect involves phosphosensory/phosphoregulatory circuits that are auniversal feature of most bacteria and play a key role in virulence[29].

Analysis of PEG15-20 (Sigma) showed that it was not a pure tri-blockpolymer but rather a mixture of polymers of varying molecular weightsincluding ABA triblock, AB diblock and homopolymer poly(ethylene glycol)structures (FIG. 7 ). In contrast to these impure mixtures of polymersof differing molecular weights, disclosed herein is the de novosynthesis of polymers with a well-defined ABA structure, thephosphorylation of which yielded compounds with a defined number ofphosphorus atoms (phosphate groups). Results demonstrated highlyeffective anti-virulence properties of the synthesized phosphorylatedpolymers with defined ABA structure and phosphate content against themodel opportunistic pathogen Pseudomonas aeruginosa.

The disclosure will be more fully understood by reference to thefollowing examples, which detail exemplary embodiments of thedisclosure. The examples should not, however, be construed as limitingthe scope of the disclosure.

EXAMPLES Example 1

Materials and Methods.

Materials. Bisphenol A (BPA, >99%, Aldrich), naphthalene (99%, Aldrich),diphenylmethane (99%, Aldrich), and phosphorus oxychloride (POCl₃, 99%,Aldrich) were used as received. Ethylene oxide (EO, >99%, lecturebottle, Praxair) and Ethoxyethyl glycidyl ether (EEGE, 97%, Synthonix)were treated with di-n-butylmagnesium for 20 minutes, and distilled intoSchlenk flasks before use. Tetrahydrofuran (THF, HPLC, inhibitor free,Aldrich) was purified with solvent purification system (Mbraun SPS-800)and distilled from a sodium naphthalenide solution directly before use.Diphenylmethylpotassium (DPMK) was prepared as described. Initially, apotassium naphthalenide solution was prepared in dry THF with 1:4 molarratio of naphthalene to potassium. After stirring for 12 hours, 0.66mole equivalents of diphenylmethane were introduced into the solutionvia syringe, and the solution was allowed to stir at room temperaturefor at least 12 hours prior to use.

Synthesis of phosphorylated PEG-based block copolymers with ahydrophobic core. Sequential Anionic Polymerization of ABA-PEG-PEEGE. Aseries of ABA-PEG-PEEGE were synthesized by the sequential anionicpolymerization of EO and EEGE in a custom heavy-wall glass reactionflask on a Schlenk line. In a typical reaction, BPA (251 mg, 1.1 mmol)dissolved in 120 mL anhydrous THF at 0° C. under dry nitrogenatmosphere, was titrated with DPMK to form the initiator, followed byaddition of the first monomer EO (22.0 g, 500 mmol). After stirring for1 hour, the mixture was heated to 50° C. and reacted for 3 days toattain complete conversion of the EO monomer. Then, the second monomerEEGE (2.9 g, 19.8 mmol) was injected into the flask and allowed to reactfor another 3 days. The polymerization was terminated with methanol andthe polymer was recovered by precipitation in cold diethyl ether.Different chain lengths of EO and EEGE are adjusted by the feed ratio of[EO]/[Initiator] and [EEGE]/[Initiator].

Hydrolysis of ABA-PEG-PEEGE. Hydrolysis of EEGE segments of blockcopolymer was carried out in THF with 4 wt % of HCl and stirred at roomtemperature for 30 minutes. The polymers were then purified byprecipitating in cold hexane and finally dried under vacuum at 60° C. toget a yellowy wax-like product, i.e., ABA-PEG-PGly (PGly: Polyglycerol). The disappearance of peaks at 4.70 ppm (q, 1H), 1.29 ppm (d,3H), 1.19 ppm (t, 3H) in 1H-NMR confirmed the success of de-protection.

Phosphorylation of ABA-PEG-PGly. ABA-PEG-Pis (Pi: Poly phosphoric acid)were prepared by phosphorylation of ABA-PEG-PGly, which was performed ina flame-dried flask under dry nitrogen atmosphere. ABA-PEG-PGly wasdissolved in anhydrous THF at 50° C., and a ten-fold equivalent molaramount of POCl₃ was added at once via gas-tight syringe. The solutionwas stirred under nitrogen pressure for 3 hours, and then quenched bythe addition of a small amount of water. After evaporation of THF anddialysis against Milli-Q water, the sample was lyophilized to give awhite flocculent product. ³¹P-NMR (D20): 6-0.18 ppm.

Synthesis of phosphorylated PEG-based block copolymers without ahydrophobic core (PEG-Pi). The synthetic strategy of PEG-Pi is quitesimilar to that of ABA-PEG-Pi, with the exception that thepolymerization started with hydrophilic ethylene glycol instead ofhydrophobic BPA, as shown in Scheme 2. First, PEG-PEEGE was prepared bystarting with ethylene glycol. Next, polymerizing through the sequentialadding of EO and EEGE and then using the same hydrolysis process (asused to obtain PEG-PGly) and the same phosphorylation process, PEG-Piwas obtained.

Characterization of block copolymers. 1H and 31P-NMR spectra wereobtained at a Bruker Ultrashield Plus 500 MHz spectrometer andreferenced internally to solvent proton signal. Apparent molecularweights and dispersity (D) were characterized with a gel permeationchromatography (GPC) system equipped with a Waters 1515 pump, a WyattOptilab T-rEX differential refractive index (RI) detector, and a Waters2998 photodiode array (PDA) detector. For ABA-PEG-PEEGEs, ABA-PEG-PGlys,PEG-PEEGEs and PEG-PGlys, THF was used for elution at 35° C. with anelution rate of 0.8 mL/minute. Three Waters Styragel columns were usedand calibrated by polystyrene standards (Aldrich). ABA-PEG-Pis andPEG-Pis were measured in 0.1 M NaNO₃ (aq) at 25° C. with an elution rateof 1.0 mL/minute on the same setup, except three Waters Ultrahydrogelcolumns in series were used and calibrated by PEO standards (Aldrich).Regarding dispersity of a polymer, monodispersed means the polymeritself, meaning all the polymer chains have nearly identical chainlengths. For amphiphilic block copolymers in selective solvent, forexample ABA-PEG-Pi in aqueous solutions, water is a good solvent for ahydrophilic block, but a poor solvent for a hydrophobic block. In thissituation, a block copolymer can self-assemble into aggregatedstructures, such as micelles, vesicles, and the like. Sometimes,micelles/vesicles and much larger structures further aggregated frommicelles/vesicles can co-exist in the same solution.

Even for block copolymers with very narrow dispersity, under somecircumstances, the block copolymers can form non-uniform structures insolutions. Even when a block copolymer forms uniform aggregatedstructures in solution, these structures can be monodispersed, meaningall the aggregates have nearly identical size and shape.

Biological tests. Bacterial strains. Pseudomonas aeruginosa strainsMPAO1-P1 and MPAO1-P2 [16] were used in all experiments. The MPAO1-P1strain and its derivative mutant ΔPvdD were used to create the reporterconstructs, MPAO1-P1/pstS-EGFP and ΔPvdD/pstS-EGFP.

Construction of pSensor-PstS-EGFP. The promoter region of the pstS gene(P. aeruginosa MPAO1) was cloned in a pSensor vector. As noted inExample 4, PstS is the phosphate-binding component of the ABC-typetransporter complex pstSACB involved in phosphate transport into thebacterial cytoplasm. The pSensor consists of a pUCP24 vector backboneand Gateway C.1 cassette (Invitrogen) in frame with the EGFP reportergene (derived from pBI-EGFP) cloned into the Sma1 and Pst1/Hind IIIsites of the pUCP24 MCS region, respectively. The region upstream ofpstS was amplified by PCR (Platinum PCR SuperMix (Invitrogen) usingprimers PstS_F: CACCTATCCCAAAACCCCTGGTCA (SEQ ID NO:1) and PstS_R:CAAACGCTTGAGTTTCATGCCTTG (SEQ ID NO:2), and cloned into the Gatewayentry vector (pCR8/GW/Topo kit (Invitrogen)). Nucleotide sequence andorientation of the inserts were confirmed by sequencing, inserts weretransferred into pSensor vector via LR reaction using Gateway LR ClonaseII Enzyme Mix (Invitrogen). Throughout the study, vector constructs werepropagated in One Shot TOP10 Chemically Competent E. coli cells.Gentamycin (100 μg/ml) selection was used for pUCP24 and pSensor andAmpicillin (100 μg/ml) for pBI-EGFP vectors. The QIAGEN Plasmid Mini Kit(Qiagen) was used for plasmid DNA extraction.

PstS expression. P. aeruginosa MPAO1-P1/pstS-EGFP or ΔPvdD/pstS-EGFPwere grown on tryptic soy agar plates supplemented with 100 μg/mlgentamicin (Gm100) overnight. A few colonies from the overnight plateswere used to inoculate liquid TSB+Gm100 for overnight growth. Theovernight culture was used to inoculate fresh TSB+Gm100 at 1:100dilution and grown to OD_(600 nm)=0.5. Cells were pelleted bycentrifugation at 3300×g for 5 minutes, and washed twice with definedcitrate media (DCM: sodium citrate, 4.0 g/L (Sigma, S4641), (NH₄)₂SO₄,1.0 g/L (Sigma, A4915), MgSO₄·7H₂O, 0.2 g/L (Fisher, M63-50). DCM mediumis limited in both phosphate and iron. Potassium phosphate buffer, pH6.0 (PPB), was used for phosphate supplementation. The supplementationof DCM with PPB 0.1 mM was defined for phosphate limitation (DCM-Pi0.1),and with PPB 25 mM for phosphate abundance (DCM-Pi25). Washed cells wereresuspended in DCM-Pi0.1+Gm100 or DCM-Pi25+Gm100, respectively, andgrown overnight. In experiments carried out to test the phosphorylatedpolymers, bacterial cells were washed in DCM-Pi0.1 and resuspended inDCM-Pi0.1+Gm100 supplemented with 2 mM ABA-PEG-Pis or ABA-PEG-PGlys andadjusted to pH 6.0 with KOH. After overnight growth, fluorescence(excitation 485/10, emission 528/20) and absorbance (600 nm) weremeasured with a FLx800 fluorescent reader (Biotek Instruments).Fluorescence readings were normalized to absorbance. Culture conditionswere 37° C. with shaking at 180 rpm (C25 Incubator Shaker, New BrunswickScientific, Edison, N.J.).

Pyocyanin production during low-phosphate conditions. P. aeruginosaMPAO1-P2, which is known to produce higher amounts of pyocyanin thanMPAO1-P1 [16], was used in this set of experiments. The design of theexperiments was similar to the experiments described above for PstSexpression except they were performed in the absence of Gm in the DCMmedia. 2 μM Fe³⁺ (1 μM Fe₂(SO₄)₃) was added to the media in order toenhance the production of pyocyanin. Pyocyanin was extracted bychloroform followed by re-extraction in the 0.2N HCl and measured atOD₅₂₀ nm as previously described [17]. Before extraction, cell densitywas measured by the absorbance at 600 nm, and pyocyanin values werenormalized to bacterial cell density.

Pyocyanin production following exposure to virulence activating factorU-50,488, kappa opioid agonist. P. aeruginosa was known to be triggeredto express enhanced virulence when exposed to kappa opioid, host factorsknown to be released into the gut during physiologic stress [17]. P.aeruginosa MPAO1-P1, which is highly sensitive to U-50,488, was used inthese experiments.

MPAO1-P1 was grown on tryptic soy agar plates overnight, and a fewcolonies were used to inoculate liquid TSB for overnight growth.Overnight cultures were used to inoculate fresh TSB at 1:100 dilutionand allowed to grow out for 1 hour. Next, 200 μM U-50,488 (Sigma) wasadded, and growth was continued for 10 hours. Pyocyanin was extractedand measured as described above.

Pyoverdin production. P. aeruginosa MPAO1-P1 was used in theseexperiments. The design of the experiments was similar to theexperiments described above for PstS expression except they wereperformed without Gm in the DCM media. Pyoverdin was measured byfluorescence (400/10 excitation, 460/40 emission) using FLx800fluorescent reader (Biotek Instruments). Data were normalized to celldensity measured as absorbance at 600 nm.

Caenorhabditis elegans killing assays. C. elegans N2 nematodes providedby the Caenorhabditis Genetic Center (CGC), University of Minnesota,were used in these experiments. Synchronization and pre-fasting of wormswas performed by transferring them onto plain plates with kanamycin aspreviously described [3]. P. aeruginosa MPAO1-P1 was grown overnight intryptone/yeast extract medium (TY, tryptone, 10 g/L; yeast extract, 5g/L) and diluted at 1:100 in 0.1×TY (TY diluted 10-fold with water).Potassium phosphate buffer, pH 6.0, was included in the 0.1×TY to afinal concentration of 0.1 mM. After 1 hour of growth, the kappa-opioidreceptor agonist U-50,488 was added to a final concentration of 50 μMfollowed by 2 hours growth as previously described [18, 19]. Two ml ofthe microbial culture was adjusted to room temperature and poured in the30-mm-diameter dishes into which pre-fasting nematodes (10 nematodes perplate) were transferred. P. aeruginosa was grown overnight in TY mediadiluted 1:100 in either 0.1×TY or 0.1×TY containing polymers at 2 mM (or5% in selected experiments, as indicated) final concentration andadjusted to pH 5.2 with KOH. Plates were incubated at room temperature,without shaking, and mortality was defined if worms did not respond tothe touch of a platinum picker.

Statistical analyses. All data are from 3 or more replicates andpresented as the mean with standard deviation presented as error bars.Statistical analysis was performed using SigmaPlot software. In C.elegans experiments, Long-rank (Mantiel-Cox) test (GraphPad Prizm 7) wasused with significance accepted as a p-value<0.05. In in vitroexperiments, Student t-tests were used and significance determined to bep-value<0.05.

Scanning electron microscopy (SEM). P. aeruginosa MPAO1 was grown intryptic soy broth (TSB) overnight. Overnight cultures (2 ml) werecentrifuged at 6,000 rpm, 5 minutes, room temperature, and pellets weregently washed (3 times) with DCM-Pi0.1 (see description of PstSexpression, above). Washed pellets were suspended in 1 ml of DCM-Pi0.1or 2 mM ABA-PEG20k-Pi20 or 2 mM PEG20k-Pi20. ABA-PEG20k-Pi20 andPEG20k-Pi20 solutions were prepared in DCM-Pi0.1. The pH of thesephosphorylated polymers is around 2-3, therefore, the pH was adjusted byKOH to DCM-Pi0.1 (pH5.5). Bacteria were grown for 4 hours, then cellswere pelleted by centrifugation at 6,000 rpm, 5 minutes, roomtemperature, and gently washed (3 times) with phosphate-buffered saline(PBS). Bacterial cells were then dropped onto glass coverslips coatedwith poly-L-lysine. Cells were fixed in 3% glutaraldehyde buffered with0.1 M phosphate buffer, pH 7.2, washed with 0.1 M phosphate buffer, anddehydrated in a graded ethanol solution in water (30% increasedgradually to 100%; 20 minutes each). The samples were dried with a LeicaCPD300 critical point dryer and coated with Pt(80)/Pd(20) to a thicknessof 2 nm using a Cressington sputter coater, model 208HR. SEM images wereobtained using a Zeiss Merlin FE-SEM with an accelerating voltage of 1kV and a working distance of 3 mm.

Example 2

Design and Synthesis of Phosphorylated PEG-Based Block Copolymer with aHydrophobic Core (ABA-PEG-Pi).

The purpose of this study was to develop phosphate-containing PEG-basedblock copolymers with a defined ABA structure and molecular weight andto identify their effectiveness in suppressing microbial virulence usingbiological tests. The ABA structure and phosphate were shown to beinvolved in providing the biologic function of Pi-PEG 15-20. However,molecular weight measurements (FIG. 7 ) indicated that both Pi-PEG 15-20and its precursor PEG 15-20 were polydisperse, i.e., PEG 15-20 containedcomponent A, 16% of block copolymer with ABA structure, and component B,84% of block copolymer with an AB structure and a PEG homopolymer. Thephosphorylated homopolymer failed to show a protective effect inbiological tests, and separating it from the original polydispersemixture to refine the active component proved implausible, since it hasan almost identical molecular weight to the block copolymer with an ABstructure and since they both are water-soluble. As such, this complexcomposition presented challenges to determine the mechanism ofprotection of each component. Therefore, a rational design of analternative PEG with uniform composition and similar structure to theactive components in Pi-PEG 15-20 was required in order to achieve boththe key features of the ABA structure and controllable phosphatecontent.

PEG chains contain only one or two terminal hydroxyl groups suitable forfurther functionalization. To incorporate more hydroxyl groups perpolymer chain, sequential anionic copolymerization of ethylene oxide(EO) with a functional epoxide monomer, ethoxy ethyl glycidyl ether(EEGE), an ethoxyl ethylacetal protected glycidyl was used to acquireblock copolymers with polyethylene oxide as backbone, along withcontrollable hydroxyl groups [20-22]. As depicted in FIG. 1 , the designof ABA-PEG-Pi involved the initial synthesis of symmetric blockcopolymer ABA-PEG-PEEGE from Bisphenol A, followed by de-protection ofPEEGE block to recover the pendant hydroxyl groups, and the subsequentfunctionalization of all the hydroxyl groups of the block copolymer withphosphate

This strategy allowed access to a series of block copolymers withdefined ABA architecture, which consisted of three distinctive segments:(i) B group represents the small, yet very hydrophobic bis-phenol Amoiety at the polymer center, (ii) PEG blocks adjacent to thebi-aromatic center formed the inert spacer and the inner part ofhydrophilic A groups. As an integral part of the architecture, the chainlength of the PEG block played a key role in thehydrophobicity/hydrophilicity balance of the whole polymer, (iii)phosphorylated polyglycidol block acts as the outer part of hydrophilicA groups, offering biological functionality and defined phosphatecontent.

Three ABA-PEG-Pis, ABA-PEG10k-Pi10, ABA-PEG16k-Pi14 and ABA-PEG20k-Pi20were synthesized. 10 k, 16 k and 20 k corresponded to the differentmolecular weight of PEG block. By incorporating more repeating units ofphosphate (10, 14 to 20 repeating units in the above phosphorylated HMWPEGs, respectively), almost identical molar concentration of phosphatecan be maintained for each block copolymer (e.g., for 1 g of each blockcopolymer, the molar concentration of phosphate were 0.78, 0.77 and 0.80mmol, respectively, for ABA-PEG10k-Pi10, ABA-PEG16k-Pi14 andABA-PEG20k-Pi20). Initiated from bis-phenoxide, the sequential anionicring-opening polymerization of EO and EEGE was successful. This can beconfirmed by the chemical shifts seen in 1H-NMR spectra (FIG. 2A): a(δ=1.60, 6H) and b (δ=6.78, 7.09, 8H) were assigned to the dimethyl andaromatic groups of BPA, c˜h (δ=3.43-3.80) were assigned to protons ofthe main chain and lateral chains, and i (δ=4.70, 1H), j (δ=1.29, 3H)and k (δ=1.19, 3H) were ascribed to the methyl protons of the EEGEmoiety. Furthermore, the chain length of the PEG and PEEGE blocks couldbe varied by adjusting the feed ratio of EO and EEGE monomer to theinitiator BPA, and the composition of the block copolymer can bedetermined by the integrals of specific signals from each block in1H-NMR spectra. The degree of polymerization of PEEGE block (NEEGE) canbe calculated by the integration ratio:

${N_{EEGE} = {8*\frac{I_{i}}{I_{b}}}},$

where I_(i) and I_(b) are the integration of Peak i and b in FIG. 2 ,respectively.

The degree of polymerization of PEG block (N_(EG)) was given by

$N_{EG} = {2*\frac{I_{c \sim h} - {7N_{EEGE}*I_{i}}}{I_{b}}}$

where I_(c˜h) is the integration of Peak c˜h in FIG. 2 . Detailedmolecular weights characterization results for all the samples aresummarized in Table 1.

TABLE 1 List of polymers synthesized in this study. M_(a) ^(s)(kDa)M_(a) (kDa) GPC NMR D^(b) N_(EEGE) ^(c) M_(hydroxyl) ^(d) N_(phosphate)^(e) ABA-PEG-PEEGE ABA-PEG10k-E8 26.1 12.4 1.05 8.0 ABA-PEG16k-E12 31.717.8 1.07 11.6 ABA-PEG20k-E18 35.9 24.6 1.04 17.5 ABA-PEG-PGlyABA-PEG10k-G10 23.2 11.9 1.06 10.0 ABA-PEG16k-G14 27.8 17.1 1.06 13.6ABA-PEG20k-G20 31.1 23.3 1.05 19.5 ABA-PEG-PI ABA-PEG10k-P110 12.9 12.81.10 9.3 ± 0.2 ABA-PEG16k-P114 13.7 18.2 1.08 13.0 ± 0.8 ABA-PEG20k-P120 25.3 25.9 1.07 19.5 ± 0.5 

Nomenclature of the polymers: TakeABA-PEG10k-E8/ABA-PEG10k-G10/ABA-PEG10k-Pi10 as examples. 10 k is thedesigned molecular weight of PEG block; E8 means the designed repeatingunits of EEGE block is 8; G10 means the designed repeating units ofGlycerol is 10. Because hydrolysis of EEGE block released 8 alcoholgroups plus 2 primary alcohol groups at the chain ends, the total is 10repeating units for Glycerol; Pi10 indicates that the designed repeatingunits of the phosphorylated Glycerol block is 10. Other polymerdesignations disclosed herein follow this nomenclature scheme.

a: ABA-PEG-PEEGE and ABA-PEG-PGly samples were measured in THF againstPS standards; ABA-PEG-Pi samples were measured in 0.1 M NaNO₃ againstPEO standards.

b: Measured by GPC.

c: Calculated from NMR.d: N_(hydroxyl)=N_(EEGE)+2 primary alcohol groups at chain ends, NMRconfirmed the complete de-protection of EEGE repeat units.e: N_(phosphate) of ABA-PEG-Pi samples were determined by phosphoricacid titration determinations.

EEGE was chosen to be the outer block, due to the advantages that: (i)it has a similar main chain to PEG and can be co-polymerized with EOthrough an anionic mechanism, (ii) this structural similarity alsoindicates that PEG-PEEGE should be non-toxic and safe, which is relevantto the use of ABA-PEG-PEEGE in biomedical applications, and (iii) theprotective ethoxy ethylacetal groups can be easily removed by acidichydrolysis, yielding pendant hydroxyl group in each repeating unit,offering perfect functionalization sites for phosphorylation. Completehydrolysis could be verified by the disappearance of specific EEGEsignals i, j and k, comparing FIG. 2A and FIG. 2B. Finally,phosphorylation was performed by the reaction between ABA-PEG-PGlysamples with phosphorus oxychloride, which was shown to be highlyeffective. The existence of phosphate in ABA-PEG-Pi samples can beverified by the chemical shift δ=−0.18 ppm in the ³¹P-NMR spectrum (FIG.2D). The number-average molecular weights of ABA-PEG-Pi measured by GPCfor ABA-PEG10k-Pi10, ABA-PEG16k-Pi14 and ABA-PEG20k-Pi20 were 12.9 k,18.7 k and 25.8 k, respectively, and corresponded well with thosedetermined from NMR results also shown in Table 1 (12.8 k, 18.2 k and25.0 k, respectively), which further confirmed that the degree offunctionalization of the available hydroxyl groups was complete.

In order to further identify the degree of phosphorylation, phosphoricacid titration experiments were performed to identify the average numberof phosphate groups per polymer chain. Briefly, 0.1M of sodium hydroxide(NaOH) solution was titrated into the ABA-PEG-Pi/PEG-Pi solution, andthe pH changes were monitored using a pH meter with automatictemperature compensation. FIG. 2E shows a typical titration curve. ThepH value of the solution increased with the gradual addition of NaOH(left axis), two buffer region (gray column area) were observed; aftersimply taking the first derivation (right axis), two equivalence pointswere clearly visualized. The data show the characteristic behavior of adiprotic acid, and the relatively broader peaks in the buffer region wasconsistent with the behavior of a poly(phosphoric acid). These resultsare in accordance with the structure of phosphoric acid units on thepolymer chain. The average number of phosphate groups per polymer chainN_(phosphate) can be calculated by equation:

$N_{phosphate} = {{\frac{\lbrack{NaOH}\rbrack*V_{1}}{m/M_{n}}{or}N_{phosphate}} = \frac{\lbrack{NaOH}\rbrack*V_{2}}{2m/M_{n}}}$

where [NaOH] is the concentration of sodium hydroxide solution, V₁ andV₂ are the volume of sodium hydroxide solution consumed at firststitration end and second titration end, respectively. m is the mass ofABA-PEG-Pi polymer used in the titration, and M_(n) is the numberaverage molecular weight of ABA-PEG-Pi polymer. Theoretically, thevolume of NaOH solution consumed at the second titration end (V₂) shouldbe twice that found at the first titration end (V₁). In the experimentdisclosed herein, V₂ is a little bit lower than 2V₁. Without wishing tobe bound by theory, this may be due to the dissociation constantdifference between the phosphoric acid units at the chain ends and thosefar from the chain ends.

Through the above method, the average number of phosphate groups perpolymer chain N_(phosphate) for ABA-PEG10K-Pi10, ABA-PEG16K-Pi14 andABA-PEG20k-Pi20 were determined to be 9.8±0.2, 13.0±0.8, and 19.5±0.5,respectively. These results confirm that phosphorylation of availablehydroxyl groups was complete.

Example 3

Synthesis of Phosphorylated PEG-Based Block Copolymer withoutHydrophobic Core (PEG-Pi).

In order to demonstrate the structural importance of the hydrophobicmoiety, another phosphate-containing PEG-based block copolymer without ahydrophobic core, namely PEG-Pi, was synthesized for comparison. Theonly structural difference between PEG-Pi and ABA-PEG-Pi is the centermoiety (FIG. 10 ): for PEG-Pi, the center moiety is ethylene glycol;whereas for ABA-PEG-Pi, it is BPA. As depicted in FIG. 15 , thesynthesis of PEG-Pi started from ethylene glycol, through the samesequential anionic ring-opening polymerization of EO and EEGE,hydrolysis and phosphorylation, producing the final product PEG-Pi. Aswith ABA-PEG20k-Pi20, 20 k corresponded to the designed molecular weightof the PEG block, 20 was the designed number of repeating units ofphosphate incorporated into each chain. GPC elution curve analysesdemonstrated the uniform composition in the synthesized polymers (FIG. 8). The polymerization, hydrolysis and phosphorylation were similarlymonitored by NMR spectroscopy. The spectra are shown in FIG. 13 . a˜g(δ=3.43-3.80) are ascribed to protons of the main chain and lateralchains, h (δ=4.70, 1H), i (δ=1.29, 3H) and i (δ=1.19, 3H) are assignedto the methyl protons of the EEGE moiety. The disappearance of signalsh. i and j confirmed complete hydrolysis of the compound, and thechemical shift δ=−0.17 ppm in the 31P-NMR spectrum verified theexistence of phosphate in PEG20k-Pi20. The average number of phosphategroups per polymer chain N_(phosphate) for PEG20k-Pi20 calculated fromphosphoric acid titration determinations was 19.8±0.3, indicating nearly100% phosphorylation. The number-average molecular weights measured byGPC for PEG20k-E18, PEG20k-G20 and PEG20k-Pi20 were 36.5 k, 31.6 k and26.2 k, respectively, which are very close to the values ofABA-PEG20k-Pi20, making PEG20k-Pi20 an excellent analogue toABA-PEG20k-Pi20. Detailed molecular weight characterization results, GPCelution curves and NMR spectra are summarized in Table 2 and in FIGS. 8and 13 .

It is also important to note that, due to the use of a living anionicpolymerization technique, the dispersity (D) of all these PEG-basedblock copolymers were kept narrow (<1.10). Significant broadening of thecorresponding GPC traces was not observed even after de-protection andphosphorylation (FIG. 8 ), indicating excellent control over molecularweight, architecture and the number of phosphate units that were desiredfor biological tests.

Example 4

ABA-PEG-Pis Inhibit Phosphate Signaling in P. aeruginosa UnderPhosphate-Limiting Conditions.

Multiple biological tests were performed to assess the functionality ofthe synthesized polymers as anti-virulence compounds. Expression of thephosphate transport protein PstS in P. aeruginosa was used as abiomarker to determine phosphate availability of the various polymers.If PstS expression was increased, it served as a proxy indicating thatextracellular phosphate was depleted and unavailable within thephosphorylated compound. On the other hand, if PstS was observed to bedecreased, it indicated that P. aeruginosa detected sufficient phosphateavailability in the test compound. PstS is the phosphate-bindingcomponent of the ABC-type transporter complex pstSACB involved inphosphate transport into the bacterial cytoplasm. PstS is known to beinduced by phosphate limitation and suppressed in a phosphate-richextracellular environment. In order to track the expression of PstS, thepSensor-PstS-EGFP plasmid (see Example 1) was electroporated in the P.aeruginosa MPAO1-P1 strain to yield the MPAO1-P1/pstS-EGFP reporterstrain. The expression of PstS was detected by fluorescence (excitation485/10, emission 528/20) normalized to cell density measured by theabsorbance at 600 nm. As a control, PstS expression in P. aeruginosagrown in low phosphate- and high phosphate-defined citrated media (DCM)was used. Data indicated, as expected, that PstS expression wasincreased in low-phosphate medium and was nearly completely suppressedin medium containing 25 mM inorganic phosphate. All three phosphorylatedpolymers (ABA-PEG10k-Pi10, ABA-PEG16k-Pi14, and ABA-PEG20k-Pi20, 2 mM)(FIG. 3A) suppressed PstS expression indicating that Pi was availablefor bacteria. In contrast, the non-phosphorylated parent polymersABA-PEG10k-G10 (G10 means that the designed number of repeating glycerolunits is 10), ABA-PEG16k-G14, and ABA-PEG20k-G20 did not suppress PstSexpression demonstrating that there was no effect of the nascent ABAstructure on the PstS expression via some type of non-specificinteraction (FIG. 3A). Reiterative experiments were then performed thatused the ΔPvdD/pstS-EGFP strain, a pyoverdin-deficient mutant derivativeof MPAO1-P1 harboring pSensor-PstS-EGFP. By using this mutant, thedecrease in fluorescence observed with ABA-PEG-Pis was verified asattributable to decreased PstS expression and not to the production ofpyoverdin, a fluorescent compound that is also produced in this medium[23]. The pattern of PstS expression in ΔPvdD/pstS-EGFP was similar tothat observed with the MPAO1-P1/pstS-EGFP (FIG. 3B). These datademonstrate that phosphorylated polymers suppress the main signalindicating phosphate limitation, i.e., PstS expression. Phosphorylatedpolymers did not inhibit bacterial growth (FIG. 9 ).

ABA-PEG-Pis Significantly Decrease Pyocyanin Production by P. aeruginosaUnder Phosphate Limited Conditions and During Exposure to Opioids.

One of the most distinguishing features of strains of P. aeruginosa istheir production of pyocyanin, a water-soluble blue-green phenazinecompound. Pyocyanin is one of the major toxins of P. aeruginosa thatinduces rapid apoptosis of human neutrophils, and thus defines thevirulence of this highly lethal opportunistic pathogen. The productionof pyocyanin is controlled by the quorum sensing system (QS), a centralvirulence circuit in P. aeruginosa and other pathogens. The PstS-PhoBphosphate regulon, a two component membrane regulator, is activatedduring phosphate limitation and is involved in the transcriptionalactivation of QS. Thus, enrichment of media with phosphate leads tosuppression of pyocyanin production [23, 24]. Therefore, ABA-PEG-Pi wasexamined to determine whether Pi can suppress pyocyanin production in P.aeruginosa in phosphate-limited medium using DCM-Pi0.1. In this set ofexperiments, a MPAO1-P2 strain was used that produces a higher amount ofpyocyanin compared to the MPAO1-P1 strain [16]. In experiments, it wasfound that supplementation of media with iron increased pyocyaninproduction in this nutrient-limited DCM media. Therefore, DCM media wassupplemented with 2 μM Fe³⁺ (1 μM Fe₂(SO₄)₃). Results demonstrated thatboth ABA-PEG10k-Pi10 and ABA-PEG20k-Pi20 significantly decreasedpyocyanin production in P. aeruginosa MPAO1-P2 (FIG. 4A). The effect ofnon-phosphorylated compounds was significantly lower.

It has been demonstrated that endogenous opioid compounds are releasedinto the intestine during physiologic stress and induce pyocyaninproduction via the quorum sensing (QS) system of virulence activation[17, 25]. The MPAO1-P1 strain was shown to be highly responsive to thesynthetic kappa opioid U-50,488 in terms of pyocyanin production [17].Consistent with previous results, pyocyanin production was demonstratedto be significantly increased in MPAO1-P1 when exposed to 200 μM of thekappa-opioid receptor agonist U-50,488 (FIG. 4B). All three ABA-PEG-Pipolymers reduced pyocyanin at the expected background level, withABA-PEG20k-Pi20 being the most effective. The paired molecular weightnon-phosphorylated polymers were less effective in these experiments,again indicating that that the phosphate content of a polymer isrelevant to its suppressive effect on pyocyanin production.

Example 5

ABA-PEG-Pis Attenuated Animal Mortality Caused by P. aeruginosa Exposedto Opioids.

Two animal models (i.e., small animal model of Caenorhabditis elegansand mouse model) were developed to assess local phosphate depletion atsites of colonization of P. aeruginosa, and their use validated thefidelity between these models [23, 26]. The C. elegans model was used inthe experiments disclosed herein in which the opioid-induced lethalityof P. aeruginosa was shown to be suppressed by the delivery of inorganicphosphate[18]. In order to test the in vivo efficacy of the de novosynthesis of APA-PEG-Pi compounds, conditions of both opioid exposureand phosphate limitation were created. Results indicated that all threeABA-PEG-Pi polymers, at equal concentrations of 2 mM, effectivelydecreased C. elegans mortality (FIG. 6 ) with the ABA-PEG20k-Pi20displaying the greatest degree of protection. Because ABA-PEG20k-Pi20carries the highest phosphate at equal molarity, to verify that theprotective effect is not dependent on phosphate concentration,reiterative experiments were performed comparing ABA-PEG10k-Pi10 toABA-PEG20k-Pi20 at concentrations of 5 weight percent. At the sameweight concentration, ABA-PEG20k-Pi20 and ABA-PEG10k-Pi10 containednearly equal quantities of phosphate. Results demonstrated thatABA-PEG20k-Pi20 still exhibited a significantly higher protective effectcompared to ABA-PEG10k-Pi10 (FIG. 14 ), indicating that the highermolecular weight leads to a greater protective effect attributable toABA-PEG20k-Pi20 relative to ABA-PEG10k-Pi10.

The hydrophobic core BPA in ABA-PEG20k-Pi20 significantly contributed tobacterial coating and its in vivo protection against lethality. In orderto confirm that it is the unique ABA structure of ABA-PEG20k-Pi20 thatplays a significant role in its protective capacity, PEG20k-Pi20 wassynthesized. This polymer has a similar structure to ABA-PEG20k-Pi20,but lacks the hydrophobic core (FIG. 10 ). As presented in FIG. 11A,results showed that both phosphorylated polymers suppressed PstSexpression to the same degree, demonstrating that both can serve asphosphate delivery molecules. In C. elegans experiments, however, theprotective effect of amphiphilic ABA-PEG20k-Pi20 was significantlygreater than the protective effect of the hydrophilic PEG20k-Pi20molecule (FIG. 11B). Because ABA-PEG-Pis may adhere to and shield thebacterial surface, the coating capacities of ABA-PEG20k-Pi20 andPEG20k-Pi20 were examined. This was performed using scanning electronmicroscopy (SEM) on P. aeruginosa. Bacteria were cultured in differentmedia for several hours, washed with buffer solution, dried in acritical point dryer, and coated with Pt/Pd before images were taken.FIGS. 11C, 11D, and 11E display images of P. aeruginosa cultured inphosphate-limited (DCM Pi-0.1 mM) media only, cultured in DCM Pi-0.1 mMcontaining 2 mM PEG20k-Pi20 and cultured in DCM Pi-0.1 mM containing 2mM ABA-PEG20k-Pi20, respectively. SEM images showed pili-like filamentsin FIG. 11C-D (shown by arrows), while in FIG. 11E, pili-like filamentsdisappeared when bacteria were coincubated in the presence ofABA-PEG20k-Pi20. These findings indicate that motility appendages, keystructures involved in virulence, are influenced by the composition ofthe two compounds [27]. In the presence of ABA-PEG20k-Pi20, the surfaceof bacterial cells displayed a distinct rugged appearance. It isexpected that the hydrophobic linkage BPA acts as an anchor, insertingitself into the alkyl chain region of the bacterial membrane, thusfirmly attaching the ABA-PEG20k-Pi20 polymer to the bacterial cellsurface. In this way, amphiphilic block copolymers like ABA-PEG20k-Pi20are expected to be advantageous as bacterial surface coating agents andhence protective in vivo.

Example 6

Oral Administration of Phosphorylated Polymers is Effective to PreventSepsis and Modulate Intestinal Homeostasis.

An experiment was conducted to assess the effects of phosphorylatedpolymers, i.e., PPi-6 and ABA-PEG20k-Pi20, delivered orally as drinkingsolutions to mice subjected to 30% hepatectomy and intestinal infectionwith the human pathogen community. More particularly, the mice weredivided into the following groups. Group 1 containedstarvation-positive, antibiotic-positive mice receiving nophosphorylated PEG but exposed to the pathogen community. Data fromthree groups of mice were analyzed. Absolute (FIG. 16A) and relative tocrypt depth (FIG. 16B) Ki67 distributions were counted on 90 cryptscumulative of 3 mice in each group. Group 2 containedstarvation-positive, antibiotic-positive mice receiving 3% PPi-6 andexposed to the pathogen community. Data from three mice were analyzed,totaling 109 crypts for Ki67 counts. Group 3 containedstarvation-positive, antibiotic-positive mice receiving 1%ABA-PEG20k-Pi20 and exposed to the pathogen community. Data from threemice were analyzed, totaling 168 crypts for Ki67 counts.

The results shown in FIGS. 16A and 16B establish that phosphorylatedpolymers (3% PPi6 and 1% ABA-PEG20k-Pi20) inhibit abnormal stem cellproliferation in cecal crypts of hepatectomized mice exposed to apathogen community. Stem cell proliferation in cecal crypts, measured byimmunostaining for the Ki67 marker, provides an accepted measure ofcrypt homeostasis. These results further demonstrated the beneficialeffects of phosphorylated polymers administrations disclosed herein. Inaddition, mice provided with drinking water containing no phosphorylatedPEG exhibited a 40% mortality rate on post-operative (hepatectomy) day2, following exposure to the pathogen community. In contrast, micedrinking water containing 3% PPi6 or water containing 1% ABA-PEG20k-Pi20exhibited 0% mortality rates (post-operative day 2) following exposureto the pathogen community. Thus, both phosphorylated polymers PPi-6 andphosphorylated PEGs promoted survival of mice.

Example 7

ABA-PEG-Pis Attenuated Collagenase Activity of CollagenolyticEnterococcus faecalis and Reduced Anastomotic Leakage Rates.

Overnight incubation of two different Enterococcus faecaliscollagenolytic strains (E2 and E27) with 2 mM of ABA-PEG20k-Pi20 led tonear complete inhibition of collagenase production in both strains (from18,000 to 1,000 and from 68,000 to 5,000 collagenase units (n=6,p<0.001)) without suppressing bacterial growth. Next, the compound wastested in a mouse model of E. faecalis—mediated anastomotic leak byproviding 1% ABA-PEG20k-Pi20 in drinking water. At post-operative day(POD) 7, anastomotic healing was assessed and the total amount of E.faecalis present at the anastomotic site and the percentage of E.faecalis expressing the collagenolytic phenotype were determined. Basedon calculated anastomotic healing scores, leak rates were decreased from80% to 20% in mice drinking 1% ABA-PEG20k-Pi20 (n=10, p<0.01). InABA-PEG20k-Pi20 drinking mice, the concentration of phosphate in distalcolon mucus was increased two-fold, the mean population of E. faecalisat the site of anastomosis was decreased 12-fold with the percentage ofcollagenolytic colonies in entire populations decreased from 20% (noABA-PEG20k-Pi20) to 1% (with ABA-PEG20k-Pi20) (n=5, p<0.05).

Sodium hexametaphosphate (PPi-6) profoundly suppresses the collagenaseproduction in gram negative strains S. marcescens and P. aeruginosa andPPi-6 supplementation in the drinking water protects mice from colonicanastomosis leakage induced by these strains. However, PPi-6 onlyslightly attenuated collagenase production in E. faecalis. As describedherein, ABA-PEG20k-Pi20 suppresses collagenase activity in E. faecalisand promotes anastomotic healing in a mouse model of anastomotic leakcaused by intestinal injection of E. faecalis.

Two groups of mice were involved in this study. All mice were subjectedto colon anastomosis followed by an enema of E. faecalis E2 at POD1. Thefirst group received water, and the second group received 1%ABA-PEG20k-Pi20 dissolved in water. At the time of sacrifice at POD7,the tissues and luminal contents were collected at the site ofanastomosis, homogenized, and used for culturing. Total Enterococcuscolonization at the anastomosis site and luminal content were determinedby culture analysis using Enterococcal selective plates (BD Difco) onwhich all Enterococcus species produced black-pigmented colonies. TheCFU count was normalized to the sample weight. The total Enterococcuspopulation was not significantly different between the two groups inboth the lumen and the tissue at the anastomotic site. Thecollagenolytic population was evaluated on Enterococcal plates coveredwith skim milk on which collagenolytic species produced black colonieswith a clearing halo surrounding the colony. The amount ofcollagenolytic colonies of E. faecalis was significantly reduced byABA-PEG20k-Pi20 in both tissue and luminal contents.

Mucus layers at anastomotic sites are enriched with phosphate in micetreated with ABA-PEG20k-Pi20. Phosphate concentration was measured inmucus layer scrapped off from 1 cm² area at the site of anastomosis atthe time of sacrifice at POD7. The concentration of phosphate wassignificantly increased in ABA-PEG20k-Pi20 group (E. faecalis group23.22±5.73 versus E. faecalis+ABA-PEG20k-Pi20, 48.42±7.633 μM, p=0.0297,n=5).

The non-phosphorylated parent polymer ABA-PEG20 k is less effectivecompared to ABA-PEG20k-Pi20. The phosphorylated polymer ABA-PEG20k-Pi20is more potent compared to its parent non-phosphorylated compoundABA-PEG20 k for suppression of collagenolytic activity of E. faecalis. Acomparative analysis was performed for ABA-PEG20k-Pi20 versus ABA-PEG20k in our mouse model and found that ABA-PEG20 k is less effectivecompared to ABA-PEG20k-Pi20. Both anastomotic leak scores and number ofcollagenolytic colonies of E. faecalis were significantly higher inABA-PEG20 k group.

The inability of PPi-6 to suppress the collagenolytic activity of E.faecalis in vitro relates to its inability in vivo to improveanastomotic healing. Orally delivered 6-mer polyphosphate (PPi-6) hasbeen recently proved as a potent protective compound in anastomotic leakinduced by gram-negative collagenolytic strains of S. marcescens and P.aeruginosa. The efficacy of this compound in vivo correlated to itsability to profoundly suppress collagenase activity in these strains invivo. However, PPi-6 does not affect the collagenolytic activity in E.faecalis, and does not promote a growth. Actually, PPi-6 atconcentrations of >2 mM even suppressed the E. faecalis growth.Respectively, in in vivo experiments, the oral PPi-6 did not improve theanastomotic healing complications caused by E. faecalis.

Preparing a bowel is a critical step in colonic surgery. In most cases,it includes mechanical cleansing and a treatment with oralnon-absorbable antibiotics. However, as it becomes evident, thisprocedure cannot guaranty the bacterial clearance, and there aremultiple evidences that bacterial pathogens such E. faecalis and P.aeruginosa are often cultured from leaking anastomosis. These bacteriawere shown to complicate anastomotic healing by degradation of collagenand thereby preventing normal extracellular matrix rearrangement that isrequired for wound healing. Given the emergence of multi-drug resistantbacteria, novel formulations are required for bowel preparation. Amongcomponents of a bowel preparation, those responsible for decrease ofbacterial collagenolytic activities should be considered. Suchcomponents are to be directed to specific bacterial phenotype ratherthan to antibiotic-like effect. For this, scientific-based approachesare required. It was recently demonstrated a potency of aphosphate-based therapy using PPi-6 to improve anastomotic healing bysuppressing collagenase activity of gram-negative pathogens. In thepresent study, it was demonstrated that a potential of yet anotherphosphate-based therapeutic compound, a phosphorylated triblockcopolymer ABA-PEG20k-Pi20 to suppress the collagenolytic activity ingram-positive bacterium Enterococcus faecalis, a pathogen tightlyassociated with anastomotic leak. The polymer ABA-PEG20k-Pi20 wassynthesized de novo and verified for its ability to coat bacteria andmake its phosphate available for delivery and virulence suppression. Inthe present study, ABA-PEG20k-Pi20 delivers phosphate to the site ofanastomosis, significantly attenuates collagenolytic populations of E.faecalis at anastomotic sites and enhances anastomotic healing. Theability of ABA-PEG20k-Pi20 to suppress collagenolytic activity iscritical for its in vivo protective effect as its non-phosphorylatedparent compound ABA-PEG20 k and inorganic polyphosphate PPi-6 that wereless effective for prevention of collagenolytic activity of E. faecaliswere also less protective to prevent anastomotic leak caused by thispathogen. In some embodiments, ABA-PEG20k-Pi20 could be combined withPPi-6 to formulate a bowel preparation against broad rangecollagenolytic pathogens.

The disclosure provided herein, including the experimental datapresented in Examples 1-7, establish that de novo synthesis ofphosphorylated PEGs, e.g., ABA-PEG-Pi polymers yield compounds thatexhibit anti-microbial function against P. aeruginosa both in vitro andin vivo, with the added benefit of allowing bacterial growth to proceednormally. It is expected that these compounds will prove efficaciousagainst microbial pathogens of the vertebrate intestine, e.g., themammalian, such as human, intestine, including P. aeruginosa and otherorganisms identified as causative agents of significant diseases, suchas GI infections and inflammations as well as sepsis.

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From the disclosure herein it will be appreciated that, althoughspecific embodiments of the disclosure have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the disclosure.

1-12. (canceled)
 13. A method of producing the triblock copolymercomprising (a) covalently attaching at least two polyethylene glycolchains to a hydrophobic core comprising a carbocyclic or heterocyclicring; (b) covalently attaching at least two phosphate groups to at leastone polyethylene glycol chain.
 14. The method of claim 13 wherein atleast two polyethylene glycol chains are each covalently attached to atleast two phosphate groups. 15-26. (canceled)